Dimer Formation of Octaprenyl-diphosphate Synthase (IspB) Is Essential for Chain Length Determination of Ubiquinone

Abstract

Ubiquinone (Q), composed of a quinone core and an isoprenoid side chain, is a key component of the respiratory chain and is an important antioxidant. In Escherichia coli, the side chain of Q-8 is synthesized by octaprenyl-diphosphate synthase, which is encoded by an essential gene, ispB. To determine how IspB regulates the length of the isoprenoid, we constructed 15 ispB mutants and expressed them inE. coli and Saccharomyces cerevisiae. The Y38A and R321V mutants produced Q-6 and Q-7, and the Y38A/R321V double mutant produced Q-5 and Q-6, indicating that these residues are involved in the determination of chain length. E. colicells (ispB::cat) harboring an Arg-321 mutant were temperature-sensitive for growth, which indicates that Arg-321 is important for thermostability of IspB. Intriguingly, E. coli cells harboring wild-type ispB and the A79Y mutant produced mainly Q-6, although the activity of the enzyme with the A79Y mutation was completely abolished. When a heterodimer of His-tagged wild-type IspB and glutathioneS-transferase-tagged IspB(A79Y) was formed, the enzyme produced a shorter length isoprenoid. These results indicate that although the A79Y mutant is functionally inactive, it can regulate activity upon forming a heterodimer with wild-type IspB, and this dimer formation is important for the determination of the isoprenoid chain length. Ubiquinone (Q), composed of a quinone core and an isoprenoid side chain, is a key component of the respiratory chain and is an important antioxidant. In Escherichia coli, the side chain of Q-8 is synthesized by octaprenyl-diphosphate synthase, which is encoded by an essential gene, ispB. To determine how IspB regulates the length of the isoprenoid, we constructed 15 ispB mutants and expressed them inE. coli and Saccharomyces cerevisiae. The Y38A and R321V mutants produced Q-6 and Q-7, and the Y38A/R321V double mutant produced Q-5 and Q-6, indicating that these residues are involved in the determination of chain length. E. colicells (ispB::cat) harboring an Arg-321 mutant were temperature-sensitive for growth, which indicates that Arg-321 is important for thermostability of IspB. Intriguingly, E. coli cells harboring wild-type ispB and the A79Y mutant produced mainly Q-6, although the activity of the enzyme with the A79Y mutation was completely abolished. When a heterodimer of His-tagged wild-type IspB and glutathioneS-transferase-tagged IspB(A79Y) was formed, the enzyme produced a shorter length isoprenoid. These results indicate that although the A79Y mutant is functionally inactive, it can regulate activity upon forming a heterodimer with wild-type IspB, and this dimer formation is important for the determination of the isoprenoid chain length. ubiquinone geranylgeranyl diphosphate farnesyl diphosphate isopentenyl diphosphate polymerase chain reaction glutathioneS-transferase nitrilotriacetic acid high pressure liquid chromatography Ubiquinone (Q)1 is an essential component of the electron transport system associated with aerobic growth and oxidative phosphorylation in many organisms. It also has been reported that ubiquinone has an important role as an antioxidant in Escherichia coli (1Søballe B. Poole R.K. Microbiology ( Read. ). 2000; 146: 787-796Crossref PubMed Scopus (84) Google Scholar),Schizosaccharomyces pombe (2Suzuki K. Okada K. Kamiya Y. Zhu X. Tanaka K. Nakagawa T. Kawamukai M. Matsuda H. J. Biochem. ( Tokyo ). 1997; 121: 496-505Crossref PubMed Scopus (80) Google Scholar, 38Uchida N. Suzuki K. Saiki R. Kainou T. Tanaka T. Matsuda H. Kawamukai M. J. Bacteriol. 2000; 182: 6933-6939Crossref PubMed Scopus (63) Google Scholar), Saccharomyces cerevisiae (3Do T.Q. Schultz J.R. Clarke C.F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7534-7539Crossref PubMed Scopus (143) Google Scholar), and mammalian cells (4Frei B. Kim M.C. Ames B.N. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4879-4883Crossref PubMed Scopus (561) Google Scholar). Furthermore, it was elegantly shown that ubiquinone (or menaquinone) accepts electrons generated by the formation of protein disulfide in E. coli(5Bader M. Muse W. Ballou D.P. Gassner C. Bardwell J.C.A. Cell. 1999; 98: 217-227Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). Thus, multiple functions of ubiquinone have been proposed. The ubiquinone biosynthetic pathway, which is comprised of 10 steps including methylation, decarboxylation, hydroxylation, and isoprenoid transfer, has been studied genetically in respiratory-deficient mutants of E. coli and S. cerevisiae (6Tzagoloff A. Dieckmann C.L. Microbiol. Rev. 1990; 54: 211-225Crossref PubMed Google Scholar). Each of these organisms has a specific isoprenoid side chain length as part of the ubiquinone molecule, e.g. Q-6 for S. cerevisiae, Q-8 for E. coli, Q-9 for rat, and Q-10 for S. pombe and human. For this reason, ubiquinone species have been used for classification in microbial taxonomy (7Collins M.D. Jones D. Microbiol. Rev. 1981; 45: 316-354Crossref PubMed Google Scholar). The length of the side chain of ubiquinone is precisely defined by the action of polyprenyl-diphosphate synthases, but not by 4-hydroxybenzoate-polyprenyl-diphosphate transferases, which catalyze the condensation of 4-hydroxybenzoate and polyprenyl diphosphate (8Okada K. Suzuki K. Kamiya Y. Zhu X. Fujisaki S. Nishimura Y. Nishino T. Nakagawa T. Kawamukai M. Matsuda H. Biochim. Biophys. Acta. 1996; 1302: 217-223Crossref PubMed Scopus (104) Google Scholar). When various polyprenyl-diphosphate synthase genes, such as the mutant GGPP synthase gene from Sulfolobus acidocaldarius, the hexaprenyl-diphosphate synthase gene (COQ1) from S. cerevisiae, the heptaprenyl-diphosphate synthase gene fromHaemophilus influenzae, the octaprenyl-diphosphate synthase gene (ispB) from E. coli, the solanesyl-diphosphate synthase gene (sdsA) fromRhodobacter capsulatus, and the decaprenyl-diphosphate synthase gene (ddsA) from Gluconobacter suboxydans, are expressed in an S. cerevisiae COQ1mutant, each transformant produced mainly Q-5, -6, -7, -8, -9, and -10, respectively (9Okada K. Kainou T. Matsuda H. Kawamukai M. FEBS Lett. 1998; 431: 241-244Crossref PubMed Scopus (72) Google Scholar). When COQ2, which encodes 4-hydroxybenzoate-hexaprenyl-diphosphate transferase in S. cerevisiae, was expressed in an E. coli ubiA mutant cell line, the transformant produce Q-8, but not Q-6 (10Suzuki K. Ueda M. Yuasa M. Nakagawa T. Kawamukai M. Matsuda H. Biosci. Biotechnol. Biochem. 1994; 58: 1814-1819Crossref PubMed Scopus (65) Google Scholar). These results indicate that prenyl-diphosphate synthase determines the chain length of ubiquinone and that 4-hydroxybenzoate-polyprenyltransferases can accept the various isoprenoid chains as a substrate. In E. coli, ispB is an essential gene, responsible for the biosynthesis of both ubiquinone and menaquinone (11Okada K. Minehira M. Zhu X. Suzuki K. Nakagawa T. Matsuda H. Kawamukai M. J. Bacteriol. 1997; 179: 3058-3060Crossref PubMed Google Scholar, 12Søballe B. Poole R.K. Microbiology ( Read. ). 1999; 145: 1817-1830Crossref PubMed Scopus (181) Google Scholar). An E. coli ubiA mutant, which does not produce Q-8, is not able to grow on a non-fermentable carbon source, but can grow on glucose (10Suzuki K. Ueda M. Yuasa M. Nakagawa T. Kawamukai M. Matsuda H. Biosci. Biotechnol. Biochem. 1994; 58: 1814-1819Crossref PubMed Scopus (65) Google Scholar). However, an E. coli ubiA − menA − mutant, which lacks both ubiquinone and menaquinone biosynthesis genes, can grow only when a small amount of Q-8 is still produced by leakiness of the mutations. Thus,ubiA − menA − mutants with an absolute lack of production of Q-8 and menaquinone-8 cannot be isolated. Long-chain polyprenyl-diphosphate synthases (C40, C45, and C50) catalyze the condensation of FPP, which acts as a primer, and IPP to produce each prenyl diphosphate with various chain lengths. These enzymes possess seven conserved regions including two DDXXD motifs that are binding sites for the substrates in association with Mg2+ (13Koyama T. Obata S. Osabe M. Takeshita A. Yokoyama K. Uchida M. Nishino T. Ogura K. J. Biochem. ( Tokyo ). 1993; 113: 355-363Crossref PubMed Scopus (123) Google Scholar, 14Chen A. Kroon P.A. Poulter C.D. Protein Sci. 1994; 3: 600-607Crossref PubMed Scopus (222) Google Scholar). Short-chain polyprenyl-diphosphate synthases (C15 and C20), such as FPP and GGPP synthases, have been identified in organisms ranging from bacteria to mammals (15Kainou T. Kawamura K. Tanaka K. Matsuda H. Kawamukai M. Biochim. Biophys. Acta. 1999; 1437: 333-340Crossref PubMed Scopus (40) Google Scholar), and the mechanisms that determine the chain length have been reported (16Ohnuma S.-i. Hirooka K. Tsuruoka N. Yano M. Ohto C. Nakane H. Nishino T. J. Biol. Chem. 1998; 273: 26705-26713Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Site-directed mutagenesis of farnesyl-diphosphate synthases from Bacillus stearothermophilus (16Ohnuma S.-i. Hirooka K. Tsuruoka N. Yano M. Ohto C. Nakane H. Nishino T. J. Biol. Chem. 1998; 273: 26705-26713Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) and certain avian species (17Tarshis L.C. Proteau P.J. Kellogg B.A. Sacchettini J.C. Poulter C.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15018-15023Crossref PubMed Scopus (315) Google Scholar) and geranylgeranyl-diphosphate synthase from S. acidocaldarius(16Ohnuma S.-i. Hirooka K. Tsuruoka N. Yano M. Ohto C. Nakane H. Nishino T. J. Biol. Chem. 1998; 273: 26705-26713Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) was used to determine which amino acids are important for the determination of chain length of short-chain prenyl diphosphates. These amino acids were those the and an in amino acid the this and two amino acids in we reported that of for the DDXXD in decaprenyl-diphosphate synthase the enzyme to with chain K. Kainou T. Tanaka K. Nakagawa T. Matsuda H. Kawamukai M. J. Biochem. 1998; PubMed Scopus Google Scholar). Thus, the amino acid of polyprenyl-diphosphate synthases an important role in the of chain length determination K. Kainou T. Tanaka K. Nakagawa T. Matsuda H. Kawamukai M. J. Biochem. 1998; PubMed Scopus Google Scholar). polyprenyl-diphosphate synthases are to as a The polyprenyl-diphosphate synthases and from B. and Bacillus are composed of T. Ogura K. J. Bacteriol. 1997; 179: PubMed Google Scholar, N. T. Ogura K. J. Bacteriol. 1998; PubMed Google Scholar). GGPP synthase from a H. Y. Ogura K. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). However, the of polyprenyl-diphosphate synthases to be synthase from R. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar) was to a has with and the has with the protein P.A. Scholar), but the motifs are not In this we the of octaprenyl-diphosphate synthase from E. the we that IspB a that is important for the determination of isoprenoid chain length. enzymes and enzymes were from and diphosphate and were from was from were from were from E. coli and were used in the of J. T. Scholar). (ispB::cat) (11Okada K. Minehira M. Zhu X. Suzuki K. Nakagawa T. Matsuda H. Kawamukai M. J. Bacteriol. 1997; 179: 3058-3060Crossref PubMed Google Scholar), which is mutant of E. coli harboring was used as a to IspB mutants and for ubiquinone (8Okada K. Suzuki K. Kamiya Y. Zhu X. Fujisaki S. Nishimura Y. Nishino T. Nakagawa T. Kawamukai M. Matsuda H. Biochim. Biophys. Acta. 1996; 1302: 217-223Crossref PubMed Scopus (104) Google Scholar), which is the mutant of S. cerevisiae, was used for and ubiquinone The and were used as J. T. Scholar, M.D. J.R. PubMed Scopus Google Scholar). The and used in this are in and used in this J. T. coli K. Minehira M. Zhu X. Suzuki K. Nakagawa T. Matsuda H. Kawamukai M. J. Bacteriol. 1997; 179: 3058-3060Crossref PubMed Google and K. Suzuki K. Kamiya Y. Zhu X. Fujisaki S. Nishimura Y. Nishino T. Nakagawa T. Kawamukai M. Matsuda H. Biochim. Biophys. Acta. 1996; 1302: 217-223Crossref PubMed Scopus (104) Google M.D. J.R. PubMed Scopus Google including ispB in K. Minehira M. Zhu X. Suzuki K. Nakagawa T. Matsuda H. Kawamukai M. J. Bacteriol. 1997; 179: 3058-3060Crossref PubMed Google high high from including ispB in K. Suzuki K. Kamiya Y. Zhu X. Fujisaki S. Nishimura Y. Nishino T. Nakagawa T. Kawamukai M. Matsuda H. Biochim. Biophys. Acta. 1996; 1302: 217-223Crossref PubMed Scopus (104) Google of in K. Suzuki K. Kamiya Y. Zhu X. Fujisaki S. Nishimura Y. Nishino T. Nakagawa T. Kawamukai M. Matsuda H. Biochim. Biophys. Acta. 1996; 1302: 217-223Crossref PubMed Scopus (104) Google gene in gene in A79Y mutant ispB gene in from in in a Site-directed mutagenesis by was the of W. H. Y. ( ). PubMed Scopus Google Scholar). each and were used in which the and of was used as in was with the and in reaction and with the and in An of each of the reaction was in a to the ispB and was with the and by was with and and was coli and (ispB::cat) for of enzyme activity and ubiquinone used to ispB mutants in this of Y38A and in a E. (ispB::cat) harboring (11Okada K. Minehira M. Zhu X. Suzuki K. Nakagawa T. Matsuda H. Kawamukai M. J. Bacteriol. 1997; 179: 3058-3060Crossref PubMed Google Scholar) was with the mutant which produced that were to and The were in and on The were on and which the mutant ispB but not were and used for ubiquinone To the mutant ispB in S. gene was The and were used to the ispB gene by The was with and and (8Okada K. Suzuki K. Kamiya Y. Zhu X. Fujisaki S. Nishimura Y. Nishino T. Nakagawa T. Kawamukai M. Matsuda H. Biochim. Biophys. Acta. 1996; 1302: 217-223Crossref PubMed Scopus (104) Google Scholar), which has amino acids of the with the also was the M.D. J.R. PubMed Scopus Google Scholar). was with both by the H. Y. K. A. J. Bacteriol. PubMed Google Scholar) and was on glucose and the amino To and IspB, the to IspB were The was to The wild-type A79Y mutant ispB was in which an been to The were E. coli were to in and 10 of was of the The was for and protein was with The cells were by for 10 To His-tagged IspB, cells were in and 10 and 10 for 10 with an in an cells were for The were to a acid and for was a and with and The protein was with and To the cells were in and were by and the was with for was with 10 and and with The protein was with 10 from was which been from to to cells harboring which IspB the mutation were with and produced that were to and and were the and with and the harboring both were and Ubiquinone was by the (8Okada K. Suzuki K. Kamiya Y. Zhu X. Fujisaki S. Nishimura Y. Nishino T. Nakagawa T. Kawamukai M. Matsuda H. Biochim. Biophys. Acta. 1996; 1302: 217-223Crossref PubMed Scopus (104) Google Scholar, Biochim. Biophys. Acta. PubMed Scopus Google Scholar). The of ubiquinone was by with was on a with The ubiquinone was from the and with were and in The ubiquinone was by with as the synthase activity was by the (8Okada K. Suzuki K. Kamiya Y. Zhu X. Fujisaki S. Nishimura Y. Nishino T. Nakagawa T. Kawamukai M. Matsuda H. Biochim. Biophys. Acta. 1996; 1302: 217-223Crossref PubMed Scopus (104) Google Scholar), in which of reaction is E. coli harboring were to a in were by in and and by each with in an of the the was used as a enzyme The reaction 10 activity of FPP, and of the enzyme in a of were for such as prenyl were with and with acid H. T. Ogura K. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). The of were with and by with on the was with a The was to to the of the It is that the side chain length of ubiquinone is by the polyprenyl-diphosphate synthase (8Okada K. Suzuki K. Kamiya Y. Zhu X. Fujisaki S. Nishimura Y. Nishino T. Nakagawa T. Kawamukai M. Matsuda H. Biochim. Biophys. Acta. 1996; 1302: 217-223Crossref PubMed Scopus (104) Google Scholar), but it is not how polyprenyl-diphosphate synthase determines this length. To the of polyprenyl-diphosphate synthase, we the activity of octaprenyl-diphosphate synthase which the side chain of Q-8 in E. For this we constructed 15 IspB mutants by mutagenesis as shown in and that were Site-directed mutagenesis was the of W. H. Y. ( ). PubMed Scopus Google Scholar), and the in were by E. coli was with the mutant ispB genes, and the were used in ubiquinone has the wild-type ispB gene in the of the is to be cells harboring the mutant ispB gene produced Q-8, a of mutants produced Q-8 with small of Q-6 and not and harboring the A79Y mutant produced mainly Q-6 To the ubiquinone species produced by the of the mutant ispB gene, E. which is for the ispB gene, but in a was with the the mutant ispB which the mutant ispB and has the wild-type was as and R321V mutant were and mutant not be isolated. ispB is essential for growth of E. coli (11Okada K. Minehira M. Zhu X. Suzuki K. Nakagawa T. Matsuda H. Kawamukai M. J. Bacteriol. 1997; 179: 3058-3060Crossref PubMed Google Scholar), the to wild-type ispB with these mutants that the and mutants not The mutants that the of the wild-type gene were by ubiquinone and In the Y38A mutant, was mainly with of Q-6 and Q-8 In the mutant, and Q-6 were mainly with a Q-8 In the Y38A/R321V and R321V Q-6 was mainly with a small amount of Q-5 and and Q-8 was In the and Q-8 was mainly a was produced a that was that with wild-type IspB and These results indicate that and Arg-321 are involved in chain length of ubiquinone from harboring various mutant ispB was from E. coli harboring gene as wild-type ispB in E. coli is essential for growth, the gene of cerevisiae can be to produce a of mutant for of the (8Okada K. Suzuki K. Kamiya Y. Zhu X. Fujisaki S. Nishimura Y. Nishino T. Nakagawa T. Kawamukai M. Matsuda H. Biochim. Biophys. Acta. 1996; 1302: 217-223Crossref PubMed Scopus (104) Google Scholar). To ispB mutants and to ubiquinone production in mutant ispB with amino acids of the were was with various mutant ispB and were on glucose as a non-fermentable carbon of the on the the and mutant not indicating that these mutants not These results are with the of mutants in E. coli the ubiquinone species produced by harboring mutant ispB In the Y38A mutant, Q-8 was mainly with a amount of In the mutant, Q-8 and were mainly with a amount of Q-6 and Q-5 In the R321V mutant, ubiquinone species Q-6, and were mainly with a small amount of Q-8 In the Y38A/R321V mutant, Q-5 was the with of Q-6 to Q-8 These results that and Arg-321 are associated with chain length species of ubiquinone produced by in E. coli were to produced cerevisiae, but ubiquinone species were shorter in length in S. cerevisiae with the produced inE. ispB mutants for and two temperature-sensitive The harboring the Arg-321 mutant on and but only The growth of the mutants are shown in B. The and R321V mutant but the and mutant not grow and the R321V mutant and a These results indicate that Arg-321 is important for thermostability of IspB, as as that ispB is essential for the growth we constructed the 15 IspB mutants by mutagenesis and expressed them in E. coli and S. cerevisiae. these the A79Y mutant an in that harboring the A79Y mutant produced Q-6 the A79Y mutant in S. cerevisiae not activity To the A79Y mutant, we the wild-type and A79Y mutant IspB harboring produced with a the amino of wild-type IspB. harboring and produced and with the amino and were and activity was as and activity of octaprenyl-diphosphate synthase, but activity not that the of for the enzyme activity of IspB, which is with the results of A79Y mutant activity in by wild-type IspB and IspB(A79Y) and a were with in and to The protein was with from on the but the protein was not in the was with from on the of wild-type IspB and the A79Y mutant was also When was with in with as a protein and to the to a heterodimer with a of was not These results indicate that wild-type IspB and IspB(A79Y) can a but these cannot with the enzyme K. Kainou T. Tanaka K. Nakagawa T. Matsuda H. Kawamukai M. J. Biochem. 1998; PubMed Scopus Google Scholar). the chain of of IspB of wild-type and A79Y in diphosphate was in this that the of the of enzyme in we not the activity of the heterodimer composed of IspB and a both and constructed the which and it with the which and the was produced Q-6 and Q-8 were from and on a a The protein was in the and the protein was in the His-tagged Thus, it was that a heterodimer of wild-type IspB and IspB(A79Y) in When enzyme activity was from isoprenoid shorter diphosphate were although in harboring only diphosphate was produced These results indicate that the IspB(A79Y) mutant can as a component of IspB to produce shorter isoprenoid chains with the isoprenoid of IspB, although IspB(A79Y) does not enzyme In E. coli, the side chain length of Q-8 is by octaprenyl-diphosphate synthase (8Okada K. Suzuki K. Kamiya Y. Zhu X. Fujisaki S. Nishimura Y. Nishino T. Nakagawa T. Kawamukai M. Matsuda H. Biochim. Biophys. Acta. 1996; 1302: 217-223Crossref PubMed Scopus (104) Google Scholar). To how IspB determines the chain we constructed 15 IspB mutants and expressed them in the E. coli mutant and in the S. cerevisiae mutant and the ubiquinone species In the IspB mutants and the ubiquinone species were the as those produced by the wild-type enzyme and However, in cells the IspB mutant was the and in cells the double mutant, the of Q-6 were the mutant of IspB not have enzyme an mutation that the protein in the mutant is by an In E. coli of the ispB gene that on a with the and mutant was Furthermore, S. cerevisiae with the not grow on These results indicate that and are important residues for The of ubiquinone species synthesized in E. coli cerevisiae harboring the mutant IspB was be to the in the of two organisms it was reported that and the produced chain length by prenyl-diphosphate synthases S. T. Ogura K. J. Biochem. ( Tokyo ). PubMed Scopus Google Scholar, S. T. Ogura K. Biochem. Biophys. 1993; PubMed Scopus Google Scholar). residues the is important for chain length determination in FPP and GGPP synthases (16Ohnuma S.-i. Hirooka K. Tsuruoka N. Yano M. Ohto C. Nakane H. Nishino T. J. Biol. Chem. 1998; 273: 26705-26713Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). we that and are not important for and an important role in activity and chain length is conserved in polyprenyl-diphosphate synthases from various organisms. of the to acid in farnesyl-diphosphate synthase from S. an enzyme that synthesized diphosphate FPP the acid ( ). 1993; PubMed Scopus (72) Google Scholar). The of this in IspB with enzyme harboring the Arg-321 mutant but not grow The the from the is conserved in polyprenyl-diphosphate synthases and FPP synthases A. Kroon P.A. Poulter C.D. Protein Sci. 1994; 3: 600-607Crossref PubMed Scopus (222) Google Scholar). However, in GGPP synthases, is in the for the amino acid in and GGPP synthases is (15Kainou T. Kawamura K. Tanaka K. Matsuda H. Kawamukai M. Biochim. Biophys. Acta. 1999; 1437: 333-340Crossref PubMed Scopus (40) Google Scholar). acid of GGPP synthases that the length of the was FPP synthases and polyprenyl-diphosphate It has been reported that an FPP synthase mutant from B. stearothermophilus in which the was by activity to that of the wild-type indicating that the amino acids in the are not essential for in FPP synthase T. K. Ogura K. Obata S. Takeshita A. J. Chem. 1994; Scopus Google Scholar). These results that the role of the in FPP and GGPP synthases is from that in polyprenyl-diphosphate In the mutant, the ubiquinone a shorter isoprenoid side chain, and cells the ispB gene with this mutation not grow These results that Arg-321 is important for thermostability of IspB. temperature-sensitive Arg-321 mutant be for to determine is an essential gene in E. of IspB is the amino acid the is important for chain length determination by FPP synthases (16Ohnuma S.-i. Hirooka K. Tsuruoka N. Yano M. Ohto C. Nakane H. Nishino T. J. Biol. Chem. 1998; 273: 26705-26713Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) and decaprenyl-diphosphate synthase K. Kainou T. Tanaka K. Nakagawa T. Matsuda H. Kawamukai M. J. Biochem. 1998; PubMed Scopus Google Scholar). In FPP synthases, the is an amino When this amino acid is with an isoprenoid the wild-type is produced (16Ohnuma S.-i. Hirooka K. Tsuruoka N. Yano M. Ohto C. Nakane H. Nishino T. J. Biol. Chem. 1998; 273: 26705-26713Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). of the amino acid in in production of a small amount of diphosphate that was the diphosphate K. Kainou T. Tanaka K. Nakagawa T. Matsuda H. Kawamukai M. J. Biochem. 1998; PubMed Scopus Google the mutant of is with that the IspB(A79Y) mutant not have enzyme wild-type E. coli harboring the A79Y mutant produced Q-6, although the A79Y mutant activity have shown that the IspB(A79Y) protein a dimer with wild-type IspB produced by the ispB gene and a ubiquinone with an chain length. have also shown that and a heterodimer and produce shorter shown in a the of the A79Y mutant not have but the heterodimer of wild-type IspB and the A79Y mutant have activity and produced a shorter isoprenoid a of wild-type IspB. in a that in the of we have produced a hexaprenyl-diphosphate Furthermore, this can be of as an a heterodimer of the polyprenyl-diphosphate synthases, such as and heptaprenyl-diphosphate synthases from M. B. T. Ogura K. J. Bacteriol. 1997; 179: PubMed Google Scholar, N. T. Ogura K. J. Bacteriol. 1998; PubMed Google Scholar). These are composed of and component does not component has conserved to polyprenyl-diphosphate the for a of and has an important role in chain length determination H. T. 1999; PubMed Scopus Google Scholar). IspB(A79Y) a role to that of component in a In of this it has been reported that the of FPP synthase with each to a in the T. Y. Nishino T. 2000; PubMed Scopus Google Scholar). The formation of of prenyl-diphosphate synthase was for the in this In of prenyl-diphosphate synthase was to be important for enzyme activity and chain length Long-chain polyprenyl-diphosphate synthase (C40, C45, and C50) have been from E. coli Fujisaki S. Nishimura Y. Nishino T. Okada K. Nakagawa T. Kawamukai M. Matsuda H. Biochem. Biophys. 1994; PubMed Scopus Google Scholar), (11Okada K. Minehira M. Zhu X. Suzuki K. Nakagawa T. Matsuda H. Kawamukai M. J. Bacteriol. 1997; 179: 3058-3060Crossref PubMed Google Scholar), R. K. Kamiya Y. Zhu X. Suzuki K. Tanaka K. Nakagawa T. Matsuda H. Kawamukai M. J. Bacteriol. 1997; 179: PubMed Google Scholar), K. Kainou T. Tanaka K. Nakagawa T. Matsuda H. Kawamukai M. J. Biochem. 1998; PubMed Scopus Google Scholar) and S. pombe (2Suzuki K. Okada K. Kamiya Y. Zhu X. Tanaka K. Nakagawa T. Kawamukai M. Matsuda H. J. Biochem. ( Tokyo ). 1997; 121: 496-505Crossref PubMed Scopus (80) Google Scholar). these synthases, for the from S. be expressed in E. coli and S. cerevisiae, we that enzymes IspB. However, the enzyme from the was not able to a in the polyprenyl-diphosphate synthase be the polyprenyl-diphosphate synthase of be an for